N-(n-acylaminoacyl)-aminoacetonitriles



Sept. 16, 1969 2 ,OOO

UNITS OF E NZYME TSUTOMU IRIKURA ETAL 3,467,691

N- (N-ACYLAMINOACYCL) -AMINOACETONITRILES Filed April 20. 1965 TENDENCY OF GLUTAMI OXALOACETIC TRANSAM$NA$E IN BLOOD SERUM FIG I ---HYPODE RMIC ADMINISTRATION OF IOO rng/lq CARBON TETRACHLORIDE (mouse) I 10 20 1 3'0 4'0 nouns A 2 TENDENCY OF GLUTAMIC PYRUVIC TRANSAMINASE IN BLOOD SERUM I '-----ADMINISTRATION OF I00 rug/kg ARBON TETRACHLORIDE mouse) --szfia a:s 22m m I WM w eza aw TETRACHI IERIBE (mouse) Tsqfornq lrjkura, Kelgo Nlsl'nno, Sezga Suzue, A Keichi Ushiyama and Hirofaka Shinada 4O INVENTORS BY r ATTORNEYS United States Patent ()1 Patented Sept. 16, 1969 3,467,691 N-(N-ACYLAMINOACYL)-AMINOACETONITRILES Tsutomu Irikul'a, 1-chome, Wakagi-cho, Itabashi-ku;

Keigo Nishino, 25 S-chome, Shimo, Kita-ku; Seigo Suzue, 35 l-chome, Minami-Senju, Arakawa-ku; and

senting a straight or branched chain alkyl radical or an aromatic radical.

The present invention is based on our discovery that secondary effects of amino acid-aminoacetonitrile can be reduced without lowering its liver protecting effects by Keichi Ushiyama, 32 3-ch0me, Ukima-cho, Kita-ku, all 5 acylation of he amino radical thereof. of Tokyo, Japan; and Hirotaka Shinada, 147 Z-chome, Some of the secondary effects of aminoacid-aminoaceto- Kita-machi, Saitama-ken, Kawagu'chi-shi, Japan nitrile will be described in more detail hereinafter.

Filed Apr- 20, 196 5, Ser- N0- 449, Aminoacetonitrile, which is the starting substance for Clalms P y, appllcatloll 1 p 22, 1964 aminoacid-aminoacetonitrile, not only has such strong Int. Cl. 4 103/30 medical effects that it completely prevents liver damage U S CL 260-465 10 Claims caused by liver damaging substances such as carbon tetrachloride, bromobenzene, thioacetoamide, etc., but it also is capable of regenerating the liver and of preventing liver ABSTRACT OF THE DISCLOSURE 15 cirrhosis. But on the other hand it has harmful pharmacodynamical effects on the vermiculation of intestines and Qompolmds that are y y strong influence both on the heart rate and the blood presn1tr1les of the formula sure, so that this substance has no value as medicine.

The aforementioned aminoacid-aminoacetonitrile has the same degree of liver protecting effect as aminoaceton1tr1le, and 1t has become possible to reduce its pharmacoln Whlch dynamical virulence to less than one-tenth and thus it has R-CO become useful as medicine. However some interesting facts have been found from is the residue of biochemical studies on these substances.

Namely, it has been found that so-called lathyrogenic R-COOH substance present in sweet pea causes denaturation NHa (liquescence) of acid muco polysaccharide which is a representing acids such as glycine, a-alanine, p-alanine, agomponeqt 25 .i i zg thus causing aminobutyric acid, a-aminoisobutyric acid, valine, leucine, fi f g an sippressmg isoleucine, norleucine, methionine, lysine, tryptophane, prolljlaga o ii q i Ce as een also 8-, 11-, 'y-, e-aminocaproic acids, aspartic acid, phenylown.t at f g n e P. t e strongest Subalanine, tyrosine, phenyl glycine, 'y-aminovaleric acid, etc. Stance m respeci 0 at yrogemc actlvltles' and represents an acyl radical, representing a The present inventors have conducted stud es for overstraight or branched chain alkyl radical or an aromatic commg the f' f hver protctms zfctlvltyi radical have been found to be useful in the treatment of pharmaco'dynamlc P lathyrogemc actlvfty, and liver diseases while having less undesirable side effects have now Succeeded m breaklng Off the correlatlon and than the parent aminoacicpaminoacetonitriles applying only the useful activities thereof to medical treatment of human diseases. 40 The above developments are tabulated as follows:

Liver protecting and Suppressing efieet on antidotal activity intestine vermiculation Lathyrogenic activity Aminoaeetonitrile Strong Str0ng Very strong. Aminoactd-aminoaeetonitrile. Equal to aminoacetonitrile 1A0 of aminoacetomtrlle Half of ammoacetonitrile. Aeyl aminoacld aminoaoetonitrile do Less than Mo oi ammoaceto- Less than oi ammoacetoof present invention. 111ml? Rimle- The present invention relates to derivatives of N-(N- It should be noted that liver cirrhosis, namely fibrinaacvlaminoacyl)-aminoacetonitrile of the formula: tion cannot be suppressed by complete removal of lathyrogenic activity and thus in case of giving priority to the liver function (for example in cases where it is desired to prevent liver disorder caused by inspirated in which r5 anesthesia, blood transfusion and so on), a lower o lathyrogenic activity is advantageous. This means the substance of the present invention is more useful in this point. Meanwhile in case of preventing liver cirrhosis 15 the resldue of tendency, aminoacid-aminoacetonitrile is more advan- R COOH tageous NH: The preventing effects, on liver disorder caused by representing amino acids such as glycine, walanine carbon tetrachloride, of acetyl glycinyl aminoacetonitrile, alanine, u-aminobutyric acid, a-aminoisobutyric acid, the slmplest one of the Substances of the present g valine, leucine, isoleucine, norleucine, methionine, lysine, tion, will be Y y of p referring to tryptophane, 5-, 11-, 6-, e-aminocaproic acids, aspartic acid, the 'P Y drawlngsphenylalanine, thyrosine, phenyl glycine, fi-amino-valeric acid, etc. and R'CO represents an acyl radical, R repre- FIG. 1 and FIG. 2 show the preventing effects, on liver disorder caused by carbon tetrachloride, of a representative substance of the present invention, acetyl glycinyl aminoacetonitrile.

The two kinds of transaminase shown in FIG. 1 and FIG. 2 will be released in a large amount into the blood serum when the parenchyma of the liver is broken. On the other hand it has been proved by various experiments that the absence of these enzymes in the blood serum indicates no disorder in the parenchyma of the liver, and thus this has been adopted as a most reliable method for the diagnosis of human liver disease.

It can be seen from FIG. 1 and FIG. 2 that acetyl glycinyl aminoacetonitrile completely prevents the liver disorder which otherwise is caused by 100 mg./kg. of carbon tetrachloride.

The substances to which the present invention relates are synthesized to N-(N-acyl aminoacid) aminoacetonitrile by acylating aminoacid-aminoacetonitrile with a suitable acylating agent, such as, for example, acid anhydride or acid halide (acid ester) etc.

This may be shown by Equation 1 as follows:

R'-CO R-CONH-CHPNC l (01' RCO-X) NH:

1|Z-CO-NH-CHr-CN NHCOR' in which RCO is the residue of NHn representing amino acid, R represents a straight or branched chain alkyl, or an aromatic radical and X represents halogen.

When acid anhydride is used as the acylating agent, the amino radical is easily acylated by reaction of aminoacid-aminoacetonitrile (or its acetate) with acid anhydride such as acetic anhydride, propionic anhydride, butyric anhydride, isobutyric anhydride and so on, for example, without a solvent or in a suitable solvent (preferably aqueous solution), so that the desired acyl aminoacid-aminoacetonitrile, N-(N-acetyl glycyl)-aminoacetonitrile, for example, is obtained.

When an acid halide, such as acetyl chloride, acetyl bromide, propionyl chloride, butyric chloride, benzoyl chloride etc., is used as the acylating agent, it is desirable that a neutralizing agent, such as sodium bicarbonate, sodium carbonate, potassium carbonate, pyridine, triethylamine, etc. be concomitantly used, and the reaction may be carried out in an aqueous solution or in an organic solvent (ether, benzene, alcohol, pyridine, for example). It is particularly advantageous in this case that the reaction takes place in an aqueous solution at a temperature between 025 C.

The present invention is hereinafter described in more detail setting forth presently preferred examples.

Aminoacid-acetonitrile [II] or a derivative thereof, which is the starting material for the present invention, is produced by a method, for example, in which acid amide synthesized from phthaloyl amino acid and aminoacetonitrile, having the general Formula III, is subjected to the action of hydrazine or its hydrate:

N-RCONHCHzCN The compound of Formula III can be obtained by combining a reactive derivative of phthaloyl amino acid, such as its halide, with aminoacetonitrile. This is shown by the following reaction scheme:

0 II C NRC OHal-HzNC H20 N C ll 0 0 II C N-RC ONHCHzCN t EXAMPLE 1 N- N-acetyl glycyl) -aminoacetonitrile 1 g. (gram) of N-glycyl-aminoacetonitrile (or its acetate) is dissolved in 5 cc. (cubic centimeters) of water, and 1.5 cc. of acetic anhydride is added at one time at room temperature (20 to 30 C.) with stirring. After 15 minutes, 1.5 cc. of acetic anhydride is again added with stirring for 30 minutes thereafter, and then condensation under diminished pressure is effected. When the resultant crystals are subjected to recrystallization from ethanol, 0.8 g. of fine plate crystals are obtained. The melting point of this substance is 157 -159 C.

Analysis.-Calculated: C, 46.44; H, 5.85; N, 27.08. Found: C, 46.41; H, 5.67; N, 27.85.

Glycyl-aminoacetonitrile used as starting material in this example can be produced as follows:

5 g. of aminoacetonitrile sulphate is dissolved in 100 cc. of water, with addition of 12 g. of sodium bicarbonate. The resulting solution is cooled to 5 C. and 50 cc. of dioxan solution of 10 g. of phthaloyl glycyl chloride is dropped thereinto with stirring for a period of 30 minutes, during which period crystals precipitate. Then stirring is continued at 5 C. for 2 hours and crystals are collected by filtration, washed with water and dried.

White needle crystals are obtained by recrystallization from acetonitrile. The melting point of this substance is between 245 C. and 246 C.

Analysis.-Calculated for C H O N N, 17.28. Found: N, 17.57.

Then 7 g. of phthaloyl glycyl aminoacetonitrile is added to 50 cc. of ethyl alcohol solution of 18 g. of 80% hydrazine hydrate and heated for 1.5 hours to distil 01f ethyl alcohol under diminished pressure. The residue is added to cc. of aqueous solution of g. of cone. hydrochloric acid and the solution is stirred for 5 minutes and cooled. Then undissolved crystals are removed and when the solution is condensed under 40 C., syrup-like residue is obtained.

The residue is dissolved in the least possible amount of methanol with addition of ethyl alcohol. When this solution is left in a refrigerator, 3 g. of white crystal of the object substance is obtained. The melting point of the substance is between 179-183 C.

Analysis.C H N Cl.Calculated: N, 28.05. Found: N, 28.87.

1 g. of glycyl-aminoacetonitrile acetate is dissolved in 5 cc. of water.'2 g. of sodium bicarbonate is added and then 1 cc. of ether solution of 0.5 g. of acetylchlorido is dropped into the solution at a temperature between and C. while stirring and cooling the solution with ice water. Stirring is continued at a temperature between 0 and 5 C. for 30 minutes thereafter, and further stirring is continued at room temperature for one hour. Then the reaction solution is filtered and when the filtrate is subjected to condensation under diminished pressure below 40 C., there are left crystals.

These crystals are extracted with hot ethanol, and undissolved substances are filtered 01f. When the filtrate is cooled, white plate crystals are obtained. The melting point of this substance is 157159 C. and the amount obtained is 0.6 g. When this substance is mixed with that obtained by Example 1, no lowering of the melting point is observed.

EXAMPLE 3 N-(N-benzoyl-glycyl)-aminoacetonitrile 1.1 g. of N-glycyl-aminoacetonitrile acetate is dissolved in cc. of water, 1.7 g. of sodium bicarbonate is added to the solution and then the solution is cooled to 0-5 C. by ice. Then 0.9 g. of benzoyl chloride is added, whereupon crystals precipitate from the solution. After four hours the precipitating crystals are collected by filtration, washed with cold water and when recrystallized with ethanol, 1.2 g. of white needle crystals are obtained. The melting point of this substance is 186-188 C.

Analysis.--Calculated: C, 60.82; H, 5.10; N, 19.35. Found: C, 60.46; H, 4.86; N, 19.53.

EXAMPLE 4 N- (N-acetyl-valyl) -aminoacetonitrile 500 mg. (milligrams) of N-valyl-aminoacetonitrile acetate is dissolved in 5 cc. of water and the solution is treated with two-time addition of 0.75 cc. of acetic anhydride in a similar way as in Example 1.'The resulting crystals are recrystallized from hot water and 420 mg. of white needle crystals having melting point between 187 and 188 C. are obtained. Infrared analysis (wave number cm.'"

v NH 3380, 3020; 1/ 0 0 1670, 1650 6NH1530,1550;6 CH 1380 5 (CH CH: 1160 In thisway the crystals are determined to be the object substance.

" EXAMPLE 5 N- (N-benzoyl-valyl) -aminoacetonitrile 4.5 g. of the object substance is obtained from 0.5 g. of N-valyl aminoacetonitrile acetate in a similar way as in Example 3. White needle crystals which are recrystallized from ethanol show a melting point between 225 and 227 C. (by capillary). When determining the melting point by micro-analysis the crsytals begin to sublimate gradually at about 200 C. and show a melting point of 234 C.

Analysis.-Calculated: C, 64.81; H, 6.61; N, 16.21. Found: C, 64.68; H, 6.41; N,-16.54.

EXAMPLE 6 N- N-benzoyl-leucyl -aminoacetonitrile 200 mg. (0.00087 mole) of N-leucyl-aminoacetonitrile acetate is dissolved in 2 cc. of water with addition of 160mg. (0.002 mole) of sodium bicarbonate, and then 1 cc. of ether solution of 210 mg. of benzoyl chloride is added at one time. When strong stirring is applied, crystals precipitate soon. Then 3 cc. of ether is added and after 30 minute stirring, crystals are collected by filtration and recrystallized from water-alcohol. White needle crystals having a melting point between 158 and 159 C. are obtained.

Analysis.-Calculated: C, 65.91; H, 7.01; N, 15.37. Found: C, 66.38; H, 6.91; N, 15.49.

EXAMPLE 7 N-(N-benzoyl-a-alanyl)-aminoacetonitrile 200 mg. of N-a-alanyl aminoacetonitrile is dissolved in 2 cc. of water, 200 mg. of sodium'bicarbonate is added thereto, and further 200 mg. of benzoyl chloride dissolved in 3 cc. of ether is added at one time. Then'strong stirring is applied at room temperature. After standing overnight, the resulting crystals are collected by filtration, and recrystallized from hot water (with active carbon), thus obtaining 210 mg. of white needle crystals.

Analysis.Calculated: C, 62.32; H, 5.67; N, 18.17. Found: C, 62.24; H, 5.37; N, 18.61.

EXAMPLE 8 N- (N-acetyl-a-alanyl)-aminoacetonitrile mg. of N-a-alanyl aminoacetonitrile acetate and 0.15 cc. of acetic anhydride are caused to react and treated in a similar way as shown inExample 1 to obtain the object substance. Recrystallized from isopropyl alcohol, 60 mg. of white needle crystals, having a melting point between 149-150 C. are obtained.

Infrared analysis (CHI-1)! NH 3380, 3300, C=O 1680, 1640 NH 1570, 1540, CH 1380, 1260 Thus the crystals are determined to be the object substance.

EXAMPLE 9 N- [N- (N-benzoyl-e-amino-caproyl) -glycy1] aminoacetonitrile Sodium bicarbonate is added to aqueous solution of N- glycyl-aminoacetonitrile acetate, and ether solution of N- benzoyl-e-amino-caproic acid is added with stirring at 5 C., stirring being continued at the same temperature for 3 hours. Then further stirring is applied at room temperature for 8 hours until the odor of the chloride disappears at room temperature, to obtain crystals. Thecrystals are recrystallized from alcohol or hot water and white needle crystals having a melting point between 158 and C are obtained.

Analysis-Calculated: N, 16.96. Found: N,17.26.

EXAMPLE 10 White needle crystals of N-(N-propionyl-glycyl)-amino-acetonitrile having a melting point between 159 and 160 C. are obtained similarly, using ethanol as recrystallization solvent.

EXAMPLE 11 White needle crystals of N (N-n-butyryl glycol)- amino-acetonitrile (M.P. 129-131 C. are similarly obtained, using ethanol.

EXAMPLE 12 White plate crystals of N-(N-n-caproyl-glycyl) aminoacetonitrile (M.P. 142l43 C.) are similarly obtained, using ethanol.

EXAMPLE 13 White plate crystals of N-(N-n-heptanoyl-glycyl)-aminoacetonitrile (M.P. 141141.5 C.) are similarly obtained, using water-ethanol.

EXAMPLE 14 White plate crystals of N-(N-n-caprylyl-glycyl)-aminoacetonitrile (M.P. 146-147 C.) are similarly obtained, using ethanol.

7 EXAMPLE White needle crystals of N-( N-acetyl-alanyl)-aminoacetonitrile (M.P. 149-150 C. are similarly obtained, using isopropyl alcohol.

EXAMPLE 16 White needle crystals of N-(N-benzoyl-alanyl)-aminoacetonitrile (M.P. 168 C) are similarly obtained, using water.

EXAMPLE 17 White needle crystals of N-(N-isobutyryl alanyl)- aminoacetonitrile (M.P. 165-166 C.) are similarly obtained, using ethanol.

EXAMPLE 18 White needle crystals of N-(N-propionyl-valyl)-aminoacetonitrile (M.P. 184-185 C.) are similarly obtained, using ethanol.

EXAMPLE 19 White needle crystals of N-(N-butyryl-valyl)-aminoacetonitrile (M.P. l72173 C.) are similarly obtained, using 30% ethanol.

EXAMPLE 20 White needle crystals of N-(N-isobutyryl-valyl)-aminoacetonitrile (M.P. 192-193 C.) are similarly obtained, using ethanol.

EXAMPLE 21 White needle crystals of N-(N-n-caproyl-valyl(-aminoacetonitrile (M.P. 167-168 C.) are similarly obtained, using ethanol.

EXAMPLE 22 White needle crystals of N-(N-n-heptanoyl valyl)- aminoacetonitrile (M.P. 167-168" C.) are obtained similarly, using ethanol.

EXAMPLE 23 White plate crystals of N-(N-n-caprylyl-valyl)-aminoacetonitrile (M.P. 148-149 C.) are similarly obtained, using water-ethanol.

EXAMPLE 24 White prism crystals of N-(N-acetyl-leucyl)-aminoacetonitrile (M.P. 144-145 C.) are similarly obtained, using acetonitrile.

EXAMPLE 25 White needle crystals of N-(N-propionyl-leucyl)-aminoacetonitrile (M.P. 127-128 C.) are similary obtained, using water.

EXAMPLE 26 White needle crystals of N-(N-isobutyryl leucyl)- aminoacetonitrile (M.P. 134-135 C. are similarly obtained, using water.

EXAMPLE 27 White needle crystals of N-(N-acetyl-fi-alanyl)-aminoacetonitrile (M.P. 143144 C.) are similarly obtained, using ethanol.

EXAMPLE 28 White plate crystals of N-(N-acetyl-e-aminocaproyl)- aminoacetonitrile (M.P. 103-104 C.) are similarly obtained, using ethyl acetate.

EXAMPLE 29 White needle crystals of N-(N-acetyl-y-aminocaproyl)- aminoacetonitrile (M.P. 147 C.) are similarly obtained, using ethyl acetate.

8 EXAMPLE 30 White needle crystals of N-(N-acetyl-fi-aminocaproyl)- aminoacetonitrile (M.P. 88-92 C.) are similarly obtained, using ethyl acetate.

EXAMPLE 31 White leaflet crystals of N-(N-acetyl-y-aminobutyryl)- aminoacetonitrile (M.P. 94-96 C.) are similarly obtained, using ethyl acetate.

EXAMPLE 32 White needle crystals of N-(N-acetyl-methionyl)- aminoacetonitrile (M.P. 109-111 C.) are similarly obtained, using isopropyl-alcohol.

EXAMPLE 33 White prism crystals of N-(N-acetyl-tryptophyl)-aminoacetonitrile (M.P. 196-l97 C.) are similarly obtained, using acetic acid.

EXAMPLE 34 White needle crystals of N-(N-acetyl-asparaginyl)- aminoacetonitrile (M.P. -196 C.) are similarly obtained, using water-ethanol.

EXAMPLE 35 White needle crystals of N-(N-ivaleryl-methionyl) aminoacetonitrile (M.P. 101-1025 C.) are similarly obtained, using water.

EXAMPLE 36 White needle crystals of N-(N-acetyl-phenylalanyl)- aminoacetonitrile (M.P. 188-189 C.) are similarly obtained, using Water-ethanol.

EXAMPLE 37 White needle crystals of N-(N-acetyl-O-acetyl-tyrosyl)- aminoacetonitrile (M.P. 179-180 C.) are similarly obtained, using water.

representing an amino acid selected from the group consisting of glycine, a-alanine, B-alanine, a-aminobutyric acid, -aminobutyric acid, a-aminoisobutyric acid, valine, leucine, isoleucine, norleucine, methionine, lysine, 18-, or, 'y-, 6-, e-aminocaproic acids, aspartic acid, phenylalanine, tyrosine, phenylglycine, a-aminovaleric acid and O-acetyltyrosine, and R'CO represents an acyl radical, R representing a member selected from the group consisting of phenyl and benzoylamino alkyl radicals, said alkyl having from 1 to 5 carbon atoms; with the proviso that where said amino acid is phenylalanine, tyrosine, phenylglycine or O-acetyltyrosine, said R represents a member selected from the group consisting of phenyl, benzoylamino alkyl,

9 10 said alkyl having from 1 to 5 carbon atoms, straight and References Cited branched chain alkyl having from 1 t0 3 carbon atoms.

2. N-(N-benzoyl-glycyl)-aminoacetonitrile. FOREIGN T P 3. N-(N-bcnzoyl-valyl)-aminoacetonitrile. 1O73288 6/1967 Great Bntam- 4. N-(N-benzoyl-leucyl)-amin0acetonitrile. 5. N-(N-benzoyl-a-alanyl)-aminoacetonitrile. 5 JOSEPH BRUST Pnmary Exammer 6. N-[N,N-benzoy1-/3-amino-caproyl)-gIycyl]-aminoace- S. T. LAWRENCE III, Assistant Examiner tonitrile.

7. N-(N-benzoyl-alanyl)-aminoacetonitrile. U.S. C1. X.R.

8. N-(N-acetyl-phenylalanyl)-aminoacetonitrile. 10 i 9. N-(N-acetyl-O-acetyI-tyrosyl)-aminoacetonitrile. 4654 424274 304 10. N (N-acctyl-phenyl-glycyl -aminoacet0nitrile. 

